Asthma is a chronic inflammatory airway disease whose prevalence is increasing worldwide and now affects 20% of children and 11% of adults in the UK. It involves a number of genetic susceptibility genes and is characterised by excessive constriction of the airways, which can be triggered by exposure to common aeroallergens, viral infections, air pollutants and even to innocuous stimuli, resulting in chest tightness, breathlessness, wheeze and cough. The disease is progressive with prolonged inflammatory damage to the airway epithelium and remodelling (structural) changes to the airway walls.
Within the airways, the epithelial cells form a physical barrier to the external environment and produce secretions that protect the airways against environmental insults. A major role of the epithelium is production of mucus secretions which protect the airways by forming a barrier which traps inhaled particulates for clearance via the mucociliary escalator. Airway secretions comprise mucins and a plethora of cytoprotective molecules including antioxidants, antiproteases and defensins that contribute to innate host defence and epithelial barrier function.
The bronchial epithelium is a pseudostratified structure comprising a layer of columnar cells with underlying basal cells that act as a physical barrier to separate the external environment and the internal milieu of the lung. Evidence of a disrupted epithelium in asthma has been observed in status asthmaticus (James et al (1998) Inflammatory mechanisms in asthma, pp 1-26. Eds: Marcel Dekker, New York) and in asthmatic sputum, which contains columnar epithelium as shed ‘creola bodies’. Furthermore, biopsies obtained from asthmatic subjects show epithelial damage (Laitinen et al. (1985). Am. Rev. Respir. Dis. 131:599-606; Montefort et al. (1992) Thorax 47:499-503) and enhanced expression of the Epidermal Growth Factor Receptor (EGFR), particularly in areas of damaged epithelium where columnar epithelial cells have been shed leaving only a basal cell layer (Puddicombe et al (2000) FASEB J. 14:1362-74).
Epithelial damage in asthma is thought to occur as a consequence of a number of extrinsic factors such as environmental stimuli and inflammatory cell products which disrupt the epithelial barrier. For example, exposure to pollutants, such as ozone or nitrogen dioxide, result in increased epithelial permeability following exposure (Bayram et al. (2002) Clin. Exp. Allergy 32:1285-92), whilst dust mite allergens, exhibiting protease activity, cause epithelial damage facilitating allergen penetration through the epithelial barrier into the underlying airway tissue leading to initiation of allergic inflammation in atopic individuals. Allergen challenges induce influxes of inflammatory cells, e.g. eosinophils and release of eosinophil basic proteins that further augment epithelial damage (Erpenbeck et al. (2003) Clin. Exp. Allergy 33:331-6). Once damaged, the epithelium in asthma shows little evidence of cell proliferation to restore barrier integrity (Demoly et al. (1994) Am. J. Respir. Crit. Care Med. 150:214-7) and expression of markers associated with growth arrest are observed (Puddicombe et al. (2003) Am. J. Respir. Cell Mol. Biol. 28:61-8). Thus a protracted epithelial repair process in asthma may contribute to the establishment of chronic inflammatory and remodelling responses and to the decline in lung function and respiratory symptoms associated with asthma. Mucus, which acts to provide epithelial protection, is produced in large quantities in asthma. However, in the face of extensive epithelial damage and loss of columnar ciliated epithelial cells, clearance of this mucus is severely reduced and results in formation of mucus plugs within the airways that can impair lung function. Thus, maintenance of the integrity of the epithelium is an important component of airways defence required to prevent epithelial damage in the face of numerous environmental insults.
It has previously been found that by culturing primary bronchial epithelial cells (BECs) obtained from the airways of asthmatics or normal BECs at an air-liquid interface, the cells can be caused to differentiate to provide a fully differentiated epithelium resembling airway epithelium in vivo (Yoshisue et al. (2004) Am. J. Respir. Cell Mol. Biol. 31:491-500). Many studies using an in vitro model system of this type have shown that interleukin-13 (IL-13) and other Th2 cytokines associated with the asthma phenotype can physically alter normal epithelial differentiation to affect the epithelial barrier and its function (Atherton et al (2003) Am. J. Physiol. Lung Cell Mol. Physiol 285:L730-L739). Exposure of epithelial cells to IL-13 during differentiation, either at an air-liquid interface or in spheroid cultures, can result in reduced transepithelial resistance, alterations in tight junctional proteins and impaired epithelial polarity (Laoukili et al. (2001) J. Clin. Invest. 108:1817-24; Kondo et al. (2002) Am. J. Respir. Cell Mol. Biol. 27:536-41). These functional alterations in the epithelial barrier induced as a consequence of Th2 cytokines have suggested the potential utility for anti-cytokine therapies for the improvement of abnormal epithelial function in the face of inflammatory and environmental insults in asthma.
Using an established air-liquid interface culture model system (Puddicombe et al. (2003) Am. J. Respir. Crit. Care Med. 167: A454), the inventors have now made the further unexpected finding that, even in the absence of addition of any Th2 cytokine or damaging agent, asthmatic epithelial cultures exhibit a significantly decreased epithelial barrier integrity as determined by measurement of transepithelial electrical resistance when compared to similar cultures of epithelial cells from non-asthmatic subjects (first reported in a poster presented at The American Thoracic Society meeting, Orlando, USA on 25th May 2004; related abstract: Puddicombe et al. (2004) Am. J. Respir. Crit. Care Med. 169:A536). Whilst barrier function measurements in cultures of bronchial epithelial cells from normal or asthmatic subjects did not significantly differ over the time course examined (see FIG. 1), at all measurement time points at 7 to 21 days from the start of culture, asthmatic bronchial epithelial cell cultures demonstrated a reduced barrier function when compared to bronchial epithelial cells from normal subjects cultured under identical conditions. Thus whilst it was previously known that environmental stimuli and inflammatory cell products will promote bronchial epithelial damage in asthmatics, it is now postulated that the extent of damage observed in asthmatic epithelium may actually be a consequence of, or contributed to by, an additional intrinsic susceptibility independent of any action of Th2 cytokines or detrimental effects of environmental or inflammatory agents. An increased epithelial permeability in asthma will likely impair epithelial barrier integrity (Puddicombe et al. (2004) Am. J. Respir. Crit. Care Med. 169:A536) and lead to greater penetration of noxious agents into the airway wall with a higher propensity for extensive epithelial damage. Enhanced epithelial permeability to damaging environmental stimuli may also contribute to the known upregulation of mucus containing secretions observed in asthma. Hence, there is now interest in determining agents which will improve or supplement defective barrier function of bronchial epithelium in asthmatics that occurs in the absence of any exogenous Th2 cytokine or other agent to reduce or prevent epithelial permeability leading to promotion of epithelial damage. Asthmatic bronchial epithelial cells differentiated in vitro in the absence of any added Th2 or proinflammatory cytokine coupled with measurement of epithelial barrier function is proposed herein as a novel model system which can be used as a preliminary in vitro screen for such agents.